Symbiosis and insect diversification: an ancient symbiont of sap-feeding insects from the bacterial phylum Bacteroidetes

Several insect groups have obligate, vertically transmitted bacterial symbionts that provision hosts with nutrients that are limiting in the diet. Some of these bacteria have been shown to descend from ancient infections. Here we show that the large group of related insects including cicadas, leafhoppers, treehoppers, spittlebugs, and planthoppers host a distinct clade of bacterial symbionts. This newly described symbiont lineage belongs to the phylum Bacteroidetes. Analyses of 16S rRNA genes indicate that the symbiont phylogeny is completely congruent with the phylogeny of insect hosts as currently known. These results support the ancient acquisition of a symbiont by a shared ancestor of these insects, dating the original infection to at least 260 million years ago. As visualized in a species of spittlebug (Cercopoidea) and in a species of sharpshooter (Cicadellinae), the symbionts have extraordinarily large cells with an elongate shape, often more than 30 mum in length; in situ hybridizations verify that these correspond to the phylum Bacteroidetes. Candidatus Sulcia muelleri is proposed as the name of the new symbiont.     

Structural specificity of the triamines sym-homospermidine and aminopropylcadaverine in stimulating growth of spermidine auxotrophs of Escherichia coli

sym-Homospermidine (HSpe) was compared with its structural isomer, aminopropylcadaverine (AP5), and the naturally-occurring triamine, spermidine (Spe), in its ability to stimulate the growth of Spe auxotrophs of Escherichia coli. HSpe was taken up by the cells, but was less effective than Spe in stimulating growth. In at least one E. coli auxotroph, HSpe was also less effective than AP5.     

The modular xylanase Xyn10A from Rhodothermus marinus is cell-attached, and its C-terminal domain has several putative homologues among cell-attached proteins within the phylum Bacteroidetes

Until recently, the function of the fifth domain of the thermostable modular xylanase Xyn10A from Rhodothermus marinus was unresolved. A putative homologue to this domain was however identified in a mannanase (Man26A) from the same microorganism which raised questions regarding a common function. An extensive search of all accessible data-bases as well as the partially sequenced genomes of R. marinus and Cytophaga hutchinsonii showed that homologues of this domain were encoded by multiple genes in microorganisms in the phylum Bacteroidetes. Moreover, the domain occurred invariably at the C-termini of proteins that were predominantly extra-cellular/cell attached. A primary structure motif of three conserved regions including structurally important glycines and a proline was also identified suggesting a conserved 3D fold. This bioinformatic evidence suggested a possible role of this domain in mediating cell attachment. To confirm this theory, R. marinus was grown, and activity assays showed that the major part of the xylanase activity was connected to whole cells. Moreover, immunocytochemical detection using a Xyn10A-specific antibody proved presence of Xyn10A on the R. marinus cell surface. In the light of this, a revision of experimental data present on both Xyn10A and Man26A was performed, and the results all indicate a cell-anchoring role of the domain, suggesting that this domain represents a novel type of module that mediates cell attachment in proteins originating from members of the phylum Bacteroidetes.     

Phylogenetic congruence of armored scale insects (Hemiptera: Diaspididae) and their primary endosymbionts from the phylum Bacteroidetes

Insects in the sap-sucking hemipteran suborder Sternorrhyncha typically harbor maternally transmitted bacteria housed in a specialized organ, the bacteriome. In three of the four superfamilies of Sternorrhyncha (Aphidoidea, Aleyrodoidea, Psylloidea), the bacteriome-associated (primary) bacterial lineage is from the class Gammaproteobacteria (phylum Proteobacteria). The fourth superfamily, Coccoidea (scale insects), has a diverse array of bacterial endosymbionts whose affinities are largely unexplored. We have amplified fragments of two bacterial ribosomal genes from each of 68 species of armored scale insects (Diaspididae). In spite of initially using primers designed for Gammaproteobacteria, we consistently amplified sequences from a different bacterial phylum: Bacteroidetes. We use these sequences (16S and 23S, 2105 total base pairs), along with previously published sequences from the armored scale hosts (elongation factor 1alpha and 28S rDNA) to investigate phylogenetic congruence between the two clades. The Bayesian tree for the bacteria is roughly congruent with that of the hosts, with 67% of nodes identical. Partition homogeneity tests found no significant difference between the host and bacterial data sets. Of thirteen Shimodaira-Hasegawa tests, comparing the original Bayesian bacterial tree to bacterial trees with incongruent clades forced to match the host tree, 12 found no significant difference. A significant difference in topology was found only when the entire host tree was compared with the entire bacterial tree. For the bacterial data set, the treelengths of the most parsimonious host trees are only 1.8-2.4% longer than that of the most parsimonious bacterial trees. The high level of congruence between the topologies indicates that these Bacteroidetes are the primary endosymbionts of armored scale insects. To investigate the phylogenetic affinities of these endosymbionts, we aligned some of their 16S rDNA sequences with other known Bacteroidetes endosymbionts and with other similar sequences identified by BLAST searches. Although the endosymbionts of armored scales are only distantly related to the endosymbionts of the other sternorrhynchan insects, they are closely related to bacteria associated with eriococcid and margarodid scale insects, to cockroach and auchenorrynchan endosymbionts (Blattabacterium and Sulcia), and to male-killing endosymbionts of ladybird beetles. We propose the name "Candidatus Uzinura diaspidicola" for the primary endosymbionts of armored scale insects.     

Distribution and abundance of macroinvertebrates within two temporary ponds

We investigated the distributions of macroinvertebrates within two temporary ponds (Spring Peeper Pond and Taylor-Ochs Pond) in central Ohio and examined what environmental factors may be driving those distributions. We sampled macroinvertebrates in Spring Peeper Pond three times from May to July 2001, and Taylor-Ochs Pond two times from May to June 2001. Macroinvertebrate distributions were significantly aggregated on all sampling dates in both ponds. Bivalve abundance in Spring Peeper Pond was higher in shallower water. The distribution of bivalves in Taylor-Ochs Pond was not correlated with any variable we measured. Dragonfly nymph abundance in Taylor-Ochs Pond decreased between the first and second sampling dates, whereas in Spring Peeper Pond no factor examined was correlated with dragonfly nymph density. Snail densities in Spring Peeper Pond were negatively related to dissolved oxygen and depth. In Taylor-Ochs Pond, snail abundance was positively related to temperature. The densities of damselfly nymphs in Spring Peeper Pond were positively related to dissolved oxygen and depth and declined across the study. In Spring Peeper Pond, hemipteran densities were negatively related to depth and increased across the study. Damselfly nymphs and hemipterans were not common enough in Taylor-Ochs to analyze. In general, the abiotic and biotic factors we examined explained relatively little (<37% in all cases) of the within pond distribution of the macroinvertebrates in our two study ponds.     

Autotrophic CO2 fixation via the reductive tricarboxylic acid cycle in different lineages within the phylum Aquificae: evidence for two ways of citrate cleavage

Autotrophic carbon fixation was characterized in representative members of the three lineages of the bacterial phylum Aquificae. Enzyme activity measurements and the detection of key genes demonstrated that Aquificae use the reductive tricarboxylic acid (TCA) cycle for autotrophic CO(2) fixation. This is the first time that strains of the Hydrogenothermaceae and 'Desulfurobacteriaceae' have been investigated for enzymes of autotrophic carbon fixation. Unexpectedly, two different mechanisms of citrate cleavage could be identified within the Aquificae. Aquificaceae use citryl-CoA synthetase and citryl-CoA lyase, whereas Hydrogenothermaceae and 'Desulfurobacteriaceae' use ATP citrate lyase. The first mechanism is likely to represent the ancestral version of the reductive TCA cycle. Sequence analyses further suggest that ATP citrate lyase formed by a gene fusion of citryl-CoA synthetase and citryl-CoA lyase and subsequently became involved in a modified version of this pathway. However, rather than having evolved within the Aquificae, our phylogenetic analyses indicate that Aquificae obtained their ATP citrate lyase through lateral gene transfer. Aquificae play an important role in biogeochemical processes in a variety of high-temperature habitats. Thus, these findings substantiate the hypothesis that autotrophic carbon fixation through the reductive TCA cycle is widespread and contributes significantly to biomass production particularly in hydrothermal habitats.     

Mechanism of aromatic amine carcinogen bypass by the Y-family polymerase,Dpo4

Bulky DNA damage inhibits DNA synthesis by replicative polymerases and often requires the action of error prone bypass polymerases. The exact mechanism governing adduct-induced mutagenesis and its dependence on the DNA sequence context remains unclear. In this work, we characterize Dpo4 binding conformations and activity with DNA templates modified with the carcinogenic DNA adducts, 2-aminofluoene (AF) or N-acetyl-2-aminofluorene (AAF), using single-molecule FRET (smFRET) analysis and DNA synthesis extension assays. We find that in the absence of dNTPs, both adducts alter polymerase binding as measured by smFRET, but the addition of dNTPs induces the formation of a ternary complex having what appears to be a conformation similar to the one observed with an unmodified DNA template. We also observe that the misincorporation pathways for each adduct present significant differences: while an AF adduct induces a structure consistent with the previously observed primer-template looped structure, its acetylated counterpart uses a different mechanism, one consistent with a dNTP-stabilized misalignment mechanism.     

Inhibition of tissue-bound semicarbazide-sensitive amine oxidase by two haloamines, 2-bromoethylamine and 3-bromopropylamine

Various mammalian tissues contain membrane-bound amine oxidase termed semicarbazide-sensitive amine oxidase (SSAO). A variety of compounds has been identified as relatively selective SSAO inhibitors, but those inhibitors currently available also inhibit monoamine oxidase (MAO). In the present study, inhibitory properties of 2-bromoethylamine (2-BEA) and 3-bromopropylamine (3-BPA) toward rat lung-bound SSAO have been studied. Regardless of preincubation, 2-BEA could not appreciably inhibit MAO-A and MAO-B activity, but 3-BPA at relatively high concentrations inhibited only MAO-B activity. 3-BPA was a competitive and reversible SSAO inhibitor with a Ki value of 17 microM regardless of preincubation. In contrast, without preincubation, 2-BEA competitively inhibited SSAO activity with the Ki value of 2.5 microM and after preincubation, the mode of inhibition changed to be noncompetitive, indicating irreversible inhibition after the preincubation. Dialysis experiments with 2-BEA-pretreated homogenate resulted in no recovery of SSAO activity even after overnight dialysis. A decreased rate of SSAO inhibition under N2 atmosphere to that obtained under O2 was produced upon preincubation of enzyme with 2-BEA, suggesting that oxidized intermediate was necessary for its inhibitory activity. Thus, 2-BEA first interacts with SSAO to form a reversible complex with a subsequent reaction, leading this complex to the covalently bound enzyme-inhibitor adduct. The data analyzed by the plot of 1/k' vs 1/2-BEA concentrations intersected on the y-axis indicate that the inhibition by 2-BEA is not mediated by a bimolecular reaction; thus it is not an affinity-labeling agent, but a suicide SSAO inhibitor. 2-BEA may be employed as a useful compound in the studying SSAO.     

Evidence for an acetoxyarylamine as the ultimate mutagenic reactive intermediate of the carcinogenic aromatic amine 2,4-diaminotoluene

2,4-Diaminotoluene (2,4-DAT) is a mutagenic and hepatocarcinogenic aromatic amine, requiring metabolic activation. We have found that the mutagenic potency of 2,4-DAT in Salmonella TA98 is similar when activated by either Aroclor-1254-induced rat primary hepatocytes or 9000 x g supernatant. Previous work has demonstrated that 2,4-DAT is activated by cytochrome P450. The present report describes an investigation of the role of acetyltransferase in 2,4-DAT activation. Substitution of TA98 with the acetyltransferase-deficient strain TA98/1,8-DNP6 resulted in an approximately 90% decrease in the mutagenic potency for 2,4-DAT using S9 activation. The newly engineered acetyltransferase-enhanced Salmonella tester strain YG1024 (TA98(pYG219] demonstrated greatly enhanced sensitivity to the mutagenicity of 2,4-DAT. Inhibition of O-acetyltransferase activity, either with the selective acetyltransferase inhibitor thiolactomycin, or by competitive inhibition with an alternative substrate for the enzyme, reduced the mutagenicity of 2,4-DAT in this acetyltransferase-enhanced bacterial strain. From these data we conclude that following 2,4-DAT activation by N-hydroxylation by cytochrome P450, the resulting hydroxylamino intermediate is further activated in the bacteria via O-acetylation to form the ultimate reactive intermediate, which is postulated to be 4-acetoxyamino-2-aminotoluene.     

Distribution and location of polycyclic aromatic hydrocarbons (PAHs) and PAH-degrading bacteria within polluted soil aggregates

A study was conducted to determine the location and distribution of PAH and PAH-degrading bacteria in different aggregate size fractions of an industrially polluted soil. The estimation of PAH-degrading bacteria using an MPN microplate technique indicated that these bacteria are most numerous in the aggregate size fractions corresponding to fine silt (2–20m) and clay(<2m) compared to larger fractions or unfractionated soil.PAH concentrations were also highest in the aggregate size fraction corresponding to fine silt. Similar results were found in a spiked soil (incubated for 6 months) with similar carbonated minerals. Transmission electron microscopy observations showed that the autochtonous PAH-degrading bacteria were embedded in the aggregates where PAHs were abundant. In spite of this extensive co-localisation PAH degradation was limited during 6 months incubation. This indicates that factors other than spatial distribution and PAH degrading ability control degradation rates. The fine silt fraction of the industrial soil had an elevated C/N ratio (35) compared to the clay fraction (C/N: 16). Thus the fraction which assumably had the highest specific surface area contained less PAH but similar numbers of PAH-degraders. N thus seem to play an important role in the long term, but as PAH degradation was low in fine size fractions, other sources/factors were probably limiting (easily degradable C, P org, O₂ etc.). Based on these findings, soil particle organization and structure of soil aggregates appear to be important for the characterization of a polluted soil (localization and sequestration). Manipulations that modify aggregation in polluted soils could thus potentially influence the accessibility and biodegradability of PAHs.     

Increased activity of spermidine N1-acetyltransferase in the adrenal cortex of rats following administration of exogenous spermidine, carbamylcholine, 2-deoxyglucose and dopamine agonists

The activity of N1-acetyltransferase was increased in the dissected adrenal cortex of the rat following a single administration of spermidine. The activity was maximal 6-8 h after the onset of treatment. The increase in enzyme activity was abolished when the rats were given cycloheximide 2 h after spermidine; this suggests that increased activity results from an augmentation in the synthesis of the enzyme. Adrenocortical spermidine N1-acetyltransferase was also induced by carbamylcholine, 2-deoxyglucose, apomorphine and piribedil, drugs that are known to cause induction of ornithine decarboxylase in that organ. Hypophysectomized rats showed reduced activity of spermidine N1-acetyltransferase when compared to sham-operated controls, and carbamylcholine no longer elicited an increase in enzyme activity in such animals. Adrenocortical spermidine N1-acetyltransferase activity of hypophysectomized rats is induced by corticotropin (ACTH). These results suggest a hormonal control over the activity of the enzyme in the adrenal cortex with ACTH acting as a mediator.     

An evaluation of the Drosophila zeste somatic eye mutation test for the detection of genotoxic azo dyes and an aromatic amine

The rate of DNA synthesis was studied in normal cell strain and in strains from patients suffering from inherited disorder ataxia telangiectasia (AT). After exposure to reactively low doses of oxic X-rays (0–4 krad) DNA synthesis was depressed in AT cell strains to a significantly lesser extent than in normal cells. This response was observed in both an “excision-deficient” and an “excision-proficient” strain. In contrast, there was no difference in DNA-synthesis inhibition between AT and normal cells after UV exposure. After X-irradiation of cells from patients with xeroderma pigmentosum, both complementation group A and XP variants, the observed rate of DNA synthesis was equal to that in normal cells. An exception was the strain XP3BR which has been shown to be X-ray sensitive. This strain exhibited diminished DNA synthesis inhibition after X-ray doses below 1 krad.These data suggest a relationship between hypersensitivity to X-rays and diminished depression of DNA synthesis.     

Distribution of two aminotransferases and D-amino acid oxidase within the nephron of young and adult rats.

In the adult rat kidney, alanine aminotransferase (EC 2.6.1.2), aspartate aminotransferase (EC 2.6.1.1) and D-amino acid oxidase (EC 1.4.3.3) were measured in glomeruli, 4 parts of the proximal tubule, 2 parts of the distal tubule and in patches from the thin limb area and the papilla. These enzymes were measured in more limited parts of the nephron during postnatal development. Adult aspartate aminotransferase activities (percentage of the highest) ranged from 100 in the distal straight segment to 25 in the late part of the proximal straight segment to 10 in the thin limb and papillary area. Alanine aminotransferase (lower by a factor of 100 in absolute terms) was distributed as the mirror image of aspartate aminotransferase within proximal and distal tubules. D-Amino acid oxidase was 850-fold higher in proximal straight segments than in medullary structures. During development alanine aminotransferase increased 6-fold and D-amino acid oxidase, 4.5-fold in proximal straight tubules but aspartate aminotransferase increased in distal straight tubles 8-fold.     

Distribution of N within Pea, Lupin, and Soybean Nodules

The ¹⁵N abundance of some, but not all, legume root nodules is significantly elevated compared to that of the whole plant. It seems probable that differences in ¹⁵N enrichment reflect differences in the assimilatory pathway of fixed N. In that context, we have determined the distribution of naturally occurring ¹⁵N in structural fractions of nodules from soybean (Glycine max L. Merr.), yellow lupin (Lupinus luteus), and pea (Pisum sativum) nodules and in chemical components from soybean nodules and to a lesser extent, pea and lupin nodules. None of the fractions of pea nodules (cortex, bacteriod, or host plant cytoplasm) was enriched in ¹⁵N. The differences among bacteriods, cortex, and plant cytoplasm were smaller in lupin than in soybean nodules, but in both, bacteriods had the highest ¹⁵N enrichment. In soybean nodules, the ¹⁵N abundance of bacteriods and cortex was higher than plant cytoplasm, but all three fractions were more enriched in ¹⁵N than the entire plant. Plant cytoplasm from soybean nodules was fractionated into protein-rich material, nonprotein alcohol precipitable material (NA), and a low molecular weight fraction. The N of the latter was further separated into N of ureides, nucleotides and free amino acids. Most of these components were either similar to or lower in ¹⁵N abundance than the plant cytoplasm as a whole, but the NA fraction showed unusual ¹⁵N enrichment. However, the percentage of nodule N in this fraction was small. NA fractions from yellow lupin and pea nodules and from soybean leaves were not enriched in ¹⁵N. Nor was the NA fraction in ruptured bacteriods and cortical tissue of soybean nodules. Variation among soybean nodule fractions in the preponderance in protein of different amino acids was not large enough to explain the differences in ¹⁵N abundances among them. A hypothesis, consistent with all known data, concerning the mechanism leading to the observed excess ¹⁵N of lupin and soybean bacteriods is offered.